Bottom Line:
Pluripotent cells can be subdivided into two distinct states, the naïve and the primed state, the latter being further advanced on the path of differentiation.Reprogramming of human stem cells into a more naïve-like state is an important research focus.The pipeline consists of identifying regulated start-ups/shut-downs in terms of molecular interactions, followed by functional annotation of the genes involved and aggregation of results across conditions, yielding sets of mechanisms that are consistently regulated in transitions towards similar states of pluripotency.

ABSTRACTPluripotent cells can be subdivided into two distinct states, the naïve and the primed state, the latter being further advanced on the path of differentiation. There are substantial differences in the regulation of pluripotency between human and mouse, and in humans only stem cells that resemble the primed state in mouse are readily available. Reprogramming of human stem cells into a more naïve-like state is an important research focus. Here, we developed a pipeline to reanalyze transcriptomics data sets that describe both states, naïve and primed pluripotency, in human and mouse. The pipeline consists of identifying regulated start-ups/shut-downs in terms of molecular interactions, followed by functional annotation of the genes involved and aggregation of results across conditions, yielding sets of mechanisms that are consistently regulated in transitions towards similar states of pluripotency. Our results suggest that one published protocol for naïve human cells gave rise to human cells that indeed share putative mechanisms with the prototypical naïve mouse pluripotent cells, such as DNA damage response and histone acetylation. However, cellular response and differentiation-related mechanisms are similar between the naïve human state and the primed mouse state, so the naïve human state did not fully reflect the naïve mouse state.

f11: Heatmap of evidence for association of comparisons and GOBP terms, for the Hanna/Gafni data set.See Figure 4 for further details.

Mentions:
We therefore compiled a combined data set from the human gene expression data that accompanied Gafni et al. [2013]10 and the mouse gene expression data from Hanna et al. [2009]8; we will refer to this and the analysis based thereon as the Hanna/Gafni data and the Hanna/Gafni analysis. In order to ensure comparability of the analyses, we restricted the Hanna/Gafni data set to the genes that were also part of the Hanna/Hanna data set. First, we performed hierarchical clustering and PCA, both visualized in panels A and B of Figure 10; Figure 2 features the equivalent analyses for the Hanna/Hanna data. Hierarchical clustering identified two major clusters in the combined data set, which correspond to the two states of pluripotency, i.e. naïve and primed, again supporting the similarity of NH and NM cells. The PCA, however, is less clear in this regard. While the first PC clearly distinguishes the PM samples from the NM samples, the human samples form a cloud around the origin of the coordinate system. This observation suggests that important gene expression changes that distinguish naïve from primed pluripotent samples in mouse are not recapitulated in human. To gain further insights into this matter, we ran our aggregation-based analysis pipeline on the combined data set. As is evident from the column-clustering in the heatmap of GOBP enrichment shown in Figure 11 (representing the same analysis stage as Figure 4; the subsequent PCA analysis is shown in Supplementary Figure S3), three of the four single comparisons making up the block aggregate naïve, cluster closely together. Indeed, their functional signatures appear to be more consistent as compared to Figure 4. Interestingly, within the list of GOBP terms that are consistently enriched in these three comparisons we find terms like stem cell maintenance, telomere maintenance, histone modification and cell cycle process, all of them known to be important for stem cell identity. Regarding the latter, pluripotent stem cells are characterized by an accelerated cell cycle, which is slowed down upon differentiation35. The mechanisms invoked in naïve human are thus similar to the naïve mouse state, and they are related to stemness. However, the fourth member of the naïve block aggregate, which corresponds to the within-species comparison of PH as source and NH as target (comparison 8), has a strikingly different biological process signature, as evident by the clustering. Since this comparison is part of the target aggregate NH, this might explain the considerable spatial distance between NH and the target aggregate NM in the PCA plot shown in Figure 12 (representing the same analysis stage as Figure 7; the underlying heatmap, taking only the significant (i.e. colored) terms of Figure 12, is shown in Supplementary Figure S4), which further points to considerable differences between NH and NM in the Hanna/Gafni analysis. On the other hand, NH and PM are located quite close to each other in the PCA plot, suggesting similarity between these, just as was the case in the Hanna/Hanna analysis (Figure 8). In line with this, our statistical assessment method again identified numerous GOBP terms that are significant for the block aggregate NHPM (Figure 13, panel C; note that panel C is truncated for the sake of readability, Supplementary Figure S2 provides an untruncated version). These terms are discussed below. Then again, there are also a number of GOBP terms that are specific for the block aggregates naïve and primed (panels A and B, respectively, of Figure 13). Among the terms for the naïve block aggregate are two terms directly related to cell cycle control, the importance of which for the pluripotent state35 was already pointed out; the respective terms are G1/S transition of mitotic cell cycle, regulation of cell cycle and associated metabolic processes. Conversely, terms related to differentiation were found for the block aggregate primed. Prominent among them were terms related to formation of ectoderm structures, such as axonogenesis and eye morphogenesis. Ectodermal differentiation-related processes were also identified as specific for the primed aggregate in the Hanna/Hanna analysis. This indicates that depletion of terms related to ectoderm differentiation is triggered by various protocols for induction of naïve pluripotency in a cross-species manner. Furthermore, within this GOBP term group are also terms that relate to mesoderm formation, like skeletal system development and kidney development, which were not observed in our Hanna/Hanna analysis (Figure 8). We take this as indication for a broader activity of differentiation processes in the primed state and, consequently, repression of these processes in the naïve state as a result of the protocol of Gafni et al. [2013]10. This is supported further by general differentiation related terms such as extracellular matrix organization and biological adhesion that are also found among the terms characterizing the block aggregate primed in the Hanna/Gafni data.

f11: Heatmap of evidence for association of comparisons and GOBP terms, for the Hanna/Gafni data set.See Figure 4 for further details.

Mentions:
We therefore compiled a combined data set from the human gene expression data that accompanied Gafni et al. [2013]10 and the mouse gene expression data from Hanna et al. [2009]8; we will refer to this and the analysis based thereon as the Hanna/Gafni data and the Hanna/Gafni analysis. In order to ensure comparability of the analyses, we restricted the Hanna/Gafni data set to the genes that were also part of the Hanna/Hanna data set. First, we performed hierarchical clustering and PCA, both visualized in panels A and B of Figure 10; Figure 2 features the equivalent analyses for the Hanna/Hanna data. Hierarchical clustering identified two major clusters in the combined data set, which correspond to the two states of pluripotency, i.e. naïve and primed, again supporting the similarity of NH and NM cells. The PCA, however, is less clear in this regard. While the first PC clearly distinguishes the PM samples from the NM samples, the human samples form a cloud around the origin of the coordinate system. This observation suggests that important gene expression changes that distinguish naïve from primed pluripotent samples in mouse are not recapitulated in human. To gain further insights into this matter, we ran our aggregation-based analysis pipeline on the combined data set. As is evident from the column-clustering in the heatmap of GOBP enrichment shown in Figure 11 (representing the same analysis stage as Figure 4; the subsequent PCA analysis is shown in Supplementary Figure S3), three of the four single comparisons making up the block aggregate naïve, cluster closely together. Indeed, their functional signatures appear to be more consistent as compared to Figure 4. Interestingly, within the list of GOBP terms that are consistently enriched in these three comparisons we find terms like stem cell maintenance, telomere maintenance, histone modification and cell cycle process, all of them known to be important for stem cell identity. Regarding the latter, pluripotent stem cells are characterized by an accelerated cell cycle, which is slowed down upon differentiation35. The mechanisms invoked in naïve human are thus similar to the naïve mouse state, and they are related to stemness. However, the fourth member of the naïve block aggregate, which corresponds to the within-species comparison of PH as source and NH as target (comparison 8), has a strikingly different biological process signature, as evident by the clustering. Since this comparison is part of the target aggregate NH, this might explain the considerable spatial distance between NH and the target aggregate NM in the PCA plot shown in Figure 12 (representing the same analysis stage as Figure 7; the underlying heatmap, taking only the significant (i.e. colored) terms of Figure 12, is shown in Supplementary Figure S4), which further points to considerable differences between NH and NM in the Hanna/Gafni analysis. On the other hand, NH and PM are located quite close to each other in the PCA plot, suggesting similarity between these, just as was the case in the Hanna/Hanna analysis (Figure 8). In line with this, our statistical assessment method again identified numerous GOBP terms that are significant for the block aggregate NHPM (Figure 13, panel C; note that panel C is truncated for the sake of readability, Supplementary Figure S2 provides an untruncated version). These terms are discussed below. Then again, there are also a number of GOBP terms that are specific for the block aggregates naïve and primed (panels A and B, respectively, of Figure 13). Among the terms for the naïve block aggregate are two terms directly related to cell cycle control, the importance of which for the pluripotent state35 was already pointed out; the respective terms are G1/S transition of mitotic cell cycle, regulation of cell cycle and associated metabolic processes. Conversely, terms related to differentiation were found for the block aggregate primed. Prominent among them were terms related to formation of ectoderm structures, such as axonogenesis and eye morphogenesis. Ectodermal differentiation-related processes were also identified as specific for the primed aggregate in the Hanna/Hanna analysis. This indicates that depletion of terms related to ectoderm differentiation is triggered by various protocols for induction of naïve pluripotency in a cross-species manner. Furthermore, within this GOBP term group are also terms that relate to mesoderm formation, like skeletal system development and kidney development, which were not observed in our Hanna/Hanna analysis (Figure 8). We take this as indication for a broader activity of differentiation processes in the primed state and, consequently, repression of these processes in the naïve state as a result of the protocol of Gafni et al. [2013]10. This is supported further by general differentiation related terms such as extracellular matrix organization and biological adhesion that are also found among the terms characterizing the block aggregate primed in the Hanna/Gafni data.

Bottom Line:
Pluripotent cells can be subdivided into two distinct states, the naïve and the primed state, the latter being further advanced on the path of differentiation.Reprogramming of human stem cells into a more naïve-like state is an important research focus.The pipeline consists of identifying regulated start-ups/shut-downs in terms of molecular interactions, followed by functional annotation of the genes involved and aggregation of results across conditions, yielding sets of mechanisms that are consistently regulated in transitions towards similar states of pluripotency.

ABSTRACTPluripotent cells can be subdivided into two distinct states, the naïve and the primed state, the latter being further advanced on the path of differentiation. There are substantial differences in the regulation of pluripotency between human and mouse, and in humans only stem cells that resemble the primed state in mouse are readily available. Reprogramming of human stem cells into a more naïve-like state is an important research focus. Here, we developed a pipeline to reanalyze transcriptomics data sets that describe both states, naïve and primed pluripotency, in human and mouse. The pipeline consists of identifying regulated start-ups/shut-downs in terms of molecular interactions, followed by functional annotation of the genes involved and aggregation of results across conditions, yielding sets of mechanisms that are consistently regulated in transitions towards similar states of pluripotency. Our results suggest that one published protocol for naïve human cells gave rise to human cells that indeed share putative mechanisms with the prototypical naïve mouse pluripotent cells, such as DNA damage response and histone acetylation. However, cellular response and differentiation-related mechanisms are similar between the naïve human state and the primed mouse state, so the naïve human state did not fully reflect the naïve mouse state.